Each Member of the Poly-r(C)-binding Protein 1 (PCBP) Family Exhibits Iron Chaperone Activity toward Ferritin

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a

Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a.     

The histone deacetylase SIRT6 suppresses the expression of the RNA-binding protein PCBP2 in glioma

More than 80% of tumors that occur in the brain are malignant gliomas. The prognosis of glioma patients is still poor, which makes glioma an urgent subject of cancer research. Previous evidence and our present data show that PCBP2 is over-expressed in human glioma tissues and predicts poor outcome. However, the mechanism by which PCBP2 is regulated in glioma remains elusive. We find that SIRT6, one of the NAD⁺-dependent class III deacetylase SIRTUINs, is down-regulated in human glioma tissues and that the level of SIRT6 is negatively correlated with PCBP2 level while H3K9ac enrichment on the promoter of PCBP2 is positively correlated with PCBP2 expression. Furthermore, we identify PCBP2 as a target of SIRT6. We demonstrate that PCBP2 expression is inhibited by SIRT6, which depends upon deacetylating H3K9ac. Finally, our results reveal that SIRT6 inhibits glioma cell proliferation and colony formation in vitro and glioma cell growth in vivo in a PCBP2 dependent manner. In summary, our findings implicate that SIRT6 inhibits PCBP2 expression through deacetylating H3K9ac and SIRT6 acts as a tumor suppressor in human glioma.     

Poly(C)-binding protein 1 (PCBP1) mediates housekeeping degradation of mitochondrial antiviral signaling (MAVS)

Mitochondrial antiviral signaling (MAVS) is a key adaptor in cellular antiviral innate immunity. We previously identified poly(C)-binding protein 2 (PCBP2) as a feedback inhibitor of MAVS that facilitates its degradation after viral infection, but little is known about the regulatory potential of poly(C)-binding protein 1 (PCBP1), which highly resembles PCBP2. Here we report that PCBP1 mediates housekeeping degradation of MAVS using the same mechanism as PCBP2 employs. Overexpression of PCBP1 impairs MAVS-mediated antiviral responses, while knockdown of PCBP1 exerts the opposite effect. The suppression is due to PCBP1-induced MAVS degradation. We observe that PCBP1 and PCBP2 show synergy in MAVS inhibition, but their expression patterns are distinct: PCBP1 is stably and abundantly expressed, while PCBP2 shows low basal expression with rapid induction after infection. Individual knockdown and subcellular fractionation analyses reveal that unlike the postinfection inhibitor PCBP2, PCBP1 continuously eliminates cellular MAVS. Our findings unravel a critical role of PCBP1 in regulating MAVS for both fine-tuning the antiviral immunity and preventing inflammation.     

The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron

BackgroundIntracellular iron transport is mediated by iron chaperone proteins known as the poly(rC)-binding proteins (PCBPs), which were originally identified as RNA/DNA-binding molecules.Scope of reviewPCBPs assume a role as not only as cytosolic iron carriers, but also as regulators of iron transport and recycling. PCBP1 is involved in the iron storage pathway that involves ferritin, while PCBP2 is involved in processes that include: iron transfer from the iron importer, divalent metal ion transporter 1; iron export mediated by ferroportin-1; and heme degradation via heme oxygenase 1.Major conclusionsBoth PCBP1 and PCBP2 possess iron-binding activity and form hetero/homo dimer complexes. These iron chaperones have a subset of non-redundant functions and regulate iron metabolism independently.General significanceThis intracellular iron chaperone system mediated by PCBPs provide a transport “gateway” of ferrous iron that may potentially link with dynamic, inter-organelle interactions to safely traffic intracellular iron.     

Identification of PCBP2, a facilitator of IRES-mediated translation, as a novel constituent of stress granules and processing bodies

Recent advances in microscopic techniques have shed light on the roles of specific subcellular structures in the regulation of gene expression. One such structure is the stress granule (SG), which is engaged in stress-triggered translational arrest by sequestering pre-initiation complexes of translation. Recent studies revealed the spatial, compositional, and functional linkage of the SG to the processing body (P-body), another cytoplasmic structure that has been implicated in mRNA degradation and siRNA- or miRNA-mediated gene silencing. In this study, we report that PCBP2, a facilitator of IRES (Internal Ribosomal Entry Site)-mediated translation, is a novel constituent of the SG and P-body. Immunofluorescence studies revealed that while PCBP2 is diffusely distributed throughout the nucleoplasm and the cytoplasm, the protein is enriched in a subset of P-bodies under normal conditions. Upon exposure to heat and arsenic stress, PCBP2 became predominantly accumulated at the SG, but was still present in Dcp1a-positive P-bodies. Live-cell imaging revealed the dynamic association of PCBP2-enriched P-bodies and the SG, and FRAP experiments demonstrated that PCBP2 actively moves in and out of the SG and P-body. Taken together, these results suggest that PCBP2 shuttles between the cytoplasm and the two structures under stress. We propose that PCBP2 may be involved in stress-induced remodeling of mRNP complexes and that it may also play a role in the rapid transition of certain silenced mRNAs into a translationally active state. Additionally, given the property of PCBP2 as a nuclear-cytoplasmic shuttling protein, PCBP2 may play a role in directly targeting nascent mRNPs to specific P-bodies for storage.     

OGFOD1, a member of the 2-oxoglutarate and iron dependent dioxygenase family, functions in ischemic signaling

The 2-oxoglutarate and iron dependent dioxygenase family are crucial for cellular adaptation to changes in oxygen concentration. We found that cells with OGFOD1 gene silencing in this family showed resistance to cell death under ischemia, and cDNA microarray analysis of OGFOD1 knockout human cells revealed downregulation of ATPAF1. Although reintroduction of the OGFOD1 wild-type gene to OGFOD1 KO cells restored ATPAF1 mRNA levels, the catalytically inactive OGFOD1 mutants did not. Furthermore, introduction of ATPAF1 gene to OGFOD1 KO cells induced ischemic cell death. Thus, OGFOD1 plays an important role in ischemic cell survival and an OGFOD1 iron binding residue is required for ATPAF1 gene expression.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

ADI1, a methionine salvage pathway enzyme,is required for Drosophila fecundity

Background

Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity.

Results

Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity.

Conclusions

In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.     

PCBP1在基因表达过程中的功能和作用机理

Poly（C）-binding protein 1 （PCBP 1）是一种RNA结合蛋白,其蛋白质相对分子质量约为38000。PCBP1含三个KH（hnRNP K homology）结构域,这些结构域对其结合RNA有重要作用。PCBP1的功能主要是参与基因转录及转录后调节,如前体mRNA的剪切、mRNA稳定性、mRNA翻译过程的沉默或增强等。本文主要对PCBP1参与的信号通路以及与人类疾病的关系进行综述,试图阐明PCBP1的生物学功能和作用机理。     

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.     

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein,Display Constitutive Activation of the Iron Starvation Response

Friedreich ataxia is a genetic disease caused by deficiencies in frataxin. This protein has homologs not only in higher eukaryotes but also in bacteria, fungi, and plants. The function of this protein is still controversial. We have identified a frataxin homolog in fission yeast, and we have analyzed whether its depletion leads to any of the phenotypes observed in other organisms. Cells deleted in pfh1 are sensitive to growth under aerobic conditions, display increased levels of total iron, hallmarks of oxidative stress such as protein carbonylation, decreased aconitase activity, and lower levels of oxygen consumption compared with wild-type cells. This mitochondrial protein seems to be important for iron and/or reactive oxygen species homeostasis. We have analyzed the proteome of cells devoid of Pfh1, and we determined that gene products up- and down-regulated upon iron depletion in wild-type cells are constitutively misregulated in this mutant. Because of the particular signaling pathway components governing the iron starvation response in fission yeast, our experiments suggest that cells lacking Pfh1 display a decrease of cytosolic available iron that triggers activation of Grx4, the common regulator of the iron starvation gene expression program. Our Schizosaccharomyces pombe Δpfh1 strain constitutes a new and useful model system to study Friedreich ataxia.